Interstrain differences in the expression and activity of Cyp2a5 in the mouse liver

Abstract

Background: Cytochrome P450 2A5 (Cyp2a5), a mouse enzyme orthologous of human CYP2A6, catalyzes a number of toxicologically important reactions, including the metabolism of nicotine, aflatoxin B1, and several other xeno- and endobiotics. Cyp2a5 expression is complex and not yet fully understood. We investigated inter-strain differences in the activity and mRNA expression of hepatic Cyp2a5. Cyp1a1/2 and Cyp2b9/10 activities were evaluated for comparative purposes. Data on the interstrain differences in the expression and activity of Cyp2a5 are important to select a suitable mouse model for studying CYP2A6-mediated metabolism.

Results: Activity of Cyp2a5 (coumarin 7-hydroxylase) was highest in DBA-2 and DBA-1, intermediate in B6D2F1 (hybrid) and low in the remaining strains (C57BL/6, C57BL/10, CBA, BALB/cAn, SW). Contrasting with the activity, background levels of Cyp2a4/5 mRNA did not differ between high- and low-activity murine strains. Phenobarbital (PB, 80 mg/kg body weight/day × 3 days, i.p.) increased Cyp2a5, Cyp1a1/2 (ethoxyresorufin-O-deethylase) and Cyp2b9/10 (bezyloxyresorufin-O-debenzylase) activities while only Cyp2a5 was enhanced by pyrazole (PYR, 100 mg/kg body weight/day × 3 days, i.p.). Inductions of Cyp2a5 activity by PYR and PB were accompanied by increases of Cyp2a4/5 mRNA. PYR and PB did not upregulate heme oxygenase-1 (hmox-1) mRNA expression in any strain, a finding that is apparently at odds with the notion that Cyp2a5 and hmox-1 inductions are coordinated events.

Conclusions: Since background levels of Cyp2a4/5 gene transcripts of high-activity strains did not differ from those of low-activity mice, distinct constitutive activities did not result from different transcription rates and/or mRNA half-lives. Results therefore suggested that interstrain differences in constitutive activity of Cyp2a5 possibly arise from distinct translation efficiencies, protein half-lives and/or enzyme kinetics toward the substrate. Data from this study indicated that all tested strains are suitable models for studying toxicants that are substrates for human CYP2A6; DBA-2, DBA-1 and the hybrid B62DF1, however, have the advantage of presenting high constitutive activities of Cyp2a5.

Keywords: Coumarin 7-hydroxylase; Cyp2a4; Cyp2a5; Heme oxygenase; Liver toxicity.

Fig. 1

Constitutive coumarin-7-hydroxylase activity (pmoles umbelliferone/mg ptn/min) in liver microsomes of different strains of mice. Histogram bar heights are mean ± SEM. Strain COH activities that differ from that of B6 strain are indicated by an asterisk above the bar (*P < 0.05, ANOVA and Dunnett’s post hoc test). N = 6 per mouse strain

Fig. 2

Constitutive levels of Cyp2a4/5 and hmox-1 mRNA in the liver of mice from different strains. a (upper panel) expression of Cyp2a4/5. b (lower panel) expression of hmox-1. Relative quantification of mRNA was made by qPCR taking C57BL/6 liver sample as the reference (*P < 0.05, Kruskal–Wallis test followed by Mann–Whitney U test with Bonferroni’s correction). N = 6 per mouse strain

Fig. 3

Pyrazole- and phenobarbital-induced expression of liver Cyp2a4/5 mRNAs in mice from different strains. Female mice were treated with phosphate buffered saline (CON, PBS 10 mL/kg body weight/day × 3 days, i.p), pyrazole (PYR, 100 mg/kg body weight/day × 3 days, i.p.) or phenobarbital (PB, 80 mg/kg body weight/day × 3 days, i.p.). Relative quantification of mRNA was made by qPCR taking the control mice (CON) as the reference. An asterisk (*) above the bar indicates that mRNA levels differ (P < 0.05, Kruskal–Wallis test followed by Mann–Whitney U test with Bonferroni’s correction) from those of vehicle-controls (CON) of the same strain. N = 6 mice of each strain per treatment group

Fig. 4

Interstrain differences in serum levels of ALT after treatment with pyrazole or phenobarbital. ALT serum levels (IU/L) in BALB/c, CBA, B6, B10, D1, D2, F1 and SW mice treated with phosphate buffered saline (CON, PBS 10 mL/kg body weight/day × 3 days, i.p), pyrazole- (PYR, 100 mg/kg body weight/day × 3 days, i.p.) or phenobarbital (PB, 80 mg/kg body weight/day × 3 days, i.p.). ALT serum levels are expressed as ratio of PYR- or PB-treated to average control group levels (100%). * levels are different from those of vehicle controls (CON) of the same strain (P < 0.05, Kruskal–Wallis test followed by Mann–Whitney U test with Bonferroni’s correction). N = 6 mice of each strain per treatment group

Fig. 5

Pyrazole and phenobarbital induced expression of liver hmox-1 mRNAs in mice from different strains. Female mice were treated with phosphate buffered saline (CON, PBS 10 mL/kg body weight/day × 3 days, i.p), pyrazole (PYR, 100 mg/kg body weight/day × 3 days, i.p.) or phenobarbital (PB, 80 mg/kg body weight/day × 3 days, i.p.). Relative quantification of mRNA was made by qPCR taking the control mice (CON) as the reference. An asterisk (*) above the bar indicates that mRNA levels differ (P < 0.05, Kruskal–Wallis test followed by Mann–Whitney U test with Bonferroni’s correction) from those of vehicle-controls (CON) of the same strain. N = 6 mice of each strain per treatment group